The overall objective of this proposal is to understand the mechanism by which a-defensin-1 inhibits HIV-1 replication. Alpha-defensins are cationic, cysteine (Cys)-rich peptides and play an important role in innate host defense. Recent studies including ours clearly demonstrate that alpha-defensin-1 has anti-HIV-1 activity. Our recent finding demonstrates that adefensin- 1 acts on the virion and the cell, although the direct effect on virions occurs under restricted conditions. We have shown that alpha-defensin-1 at a non-cytotoxic, physiological concentration can inhibit HIV-1 replication following viral entry, suggesting that alpha-defensin-1 has an effect(s) on the target cells where HIV-1 replicates. In primary CD4+ T cells, we have identified that alpha-defensin-1 inhibits protein Kinase C (PKC) signaling, a pathway important for HIV-1 infection and immune cell function. Our hypothesis is that alpha-defensin-1 has significant anti-HIV-1 activity and this activity is largely mediated through cell signaling in target cells. This proposal will study the mechanism(s) of action in primary CD4+ T cells and macrophages, the major target cells for HIV-1. In Aim 1, we will extend the post-entry study by evaluating the antiviral activity of alpha-defensin-1 against HIV-1 primary isolates. The direct effect on HIV-1 virions will be further studied using R5 viruses. We will identify the point in the virus life cycle blocked by a-defensin-1 in these primary cells. In Aim 2, we will assess the role of PKC in alpha-defensin- 1-mediated HIV-1 inhibition. Our preliminary results indicate that PKCalpha and (3 are involved in HIV-1 inhibition by alpha-defensin-1. We will assess activities of PKCalpha and (3 in primary CD4+ T cells in response to a-defensin-1. We will also evaluate the interaction of alpha-defensin-1 with PKCalpha or beta. The role of PKCalpha and beta in alpha-defensin-1-mediated anti-HIV activity will be examined by introducing constitutively active PKC. In Aim 3, we will study the contribution of structure of a-defensin-1 to the anti-HIV-1 activity. We will explore the difference in native compared to recombinant and synthetic alpha-defensin-1 in anti-HIV affect. We will define amino acid residues in a-defensin-1 important for its anti-HIV-1 activity. These studies will offer insights into the function of alpha-defensin-1 in innate immune responses against HIV-1 infection and provide an understanding of the structure-function relationship of a-defensin-1 that is essentially important in view of the efforts to develop defensin-derived peptides for prevention and therapeutic use.